Final Report Summary - NOVEL TOOLS IN PD (Novel tools for real time monitoring and quantification of protein aggregation in Parkinson s disease and related neurodegenerative disorders) Parkinson's disease (PD) is a neurodegenerative movement disorder that affects 1% of the population at 65 and ~5% at 85 years of age. The disease is characterized by the loss of dopamine producing neurons in the part of the brain that regulates movement, the substantia nigra. Compelling circumstantial evidence from pathological, genetic, animal model and biochemical studies support the hypothesis that the aggregation of the presynaptic protein α-synuclein plays a central role in the pathogenesis of PD and other neurodegenerative disease that are collectively known as synucleinopathies. However, what triggers α-syn aggregation and which form of α-syn is primary toxic entity remain unknown. This knowledge is essential for the development of effective strategies to diagnose, monitor and prevent the progression of PD. The primary objective of this project is to develop novel tools that would allow us to monitor early stages in α-syn aggregation in vivo and to determine how specific disease-associated α-syn modifications and structural changes influence α-syn properties and contribute to the development and progression of neurodegeneration in Parkinson’s disease. This knowledge is crucial for the identification of novel biomarkers, development of diagnostic tools and the identification of novel therapies based on targeting the enzymes or pathways involved in regulating these modifications. Towards achieving this goal, we have developed novel tools and chemical approaches that have enabled for the first time to produce homogeneously modified forms of α-syn, including forms that have been linked to disease pathology and progression. Using these approaches we generated a library of 24 proteins that consists of all physiologic and diseases-associated modified forms of α-syn. In addition, we were successful in identifying some of the natural enzymes responsible for regulating α-syn phosphorylation at S129, Y39 and Y125. Altogether, these advances provided unique opportunities for investigating how the presence of each or multiple post-translational modifications influences the structure, subcellular localization, and aggregation. The knowledge gained from these studies was then used to design in vivo experiments aimed at elucidating the effect of these modifications on α-syn aggregation, clearance and toxicity in neuronal and animal model of Parkinson’s disease. Our results show that some of these modifications (phosphorylation at S87 and S129 and Ubiquitination at K6 and K12) block α-syn aggregation and protect against α-syn induced toxicity. Other modifications such as N-terminal acetylation and phosphorylation at Y125 do not affect α-syn aggregation, but result in local structural perturbations near the modifications sites. Nitration of α-syn at single of multiple tyrosine residues promotes α-syn oligomerization, but significantly attenuates its fibrillization. Finally, we demonstrated that α-syn PTMs, more specifically ubiquitination and phosphorylation at S129 and Y39 play important roles in regulating the clearance of the protein via the lysosomal autophagic and/or proteasomal pathways. Collectively, our studies revealed novel insights and demonstrated that α-Syn functions under complex regulatory mechanisms involving cross-talk among different posttranslational modifications and suggest that the enzymes that regulate these modifications (e.g. Polo-like Kinase 2 [PLK2] and c-Abl) may constitute a viable therapeutic target for the treatment of PD. In addition, the identification of a specific isoform or post-translational modification pattern that correlate with PD progression could result in the identification of novel diagnostic marker and the development of novel assays for diagnosing and/or monitoring the progression of PD and related synucleinopathies. The availability of semisynthetic homogeneously post-translationally modified forms of α-syn as a substrates should aid in the development of assays to identify the natural enzymes (kinases, phosphatases, E3 ligases, deubiquitinases) involved in the regulation of these modification and will facilitate the development of quantitative assays to assess the levels of these modification during disease progression. Impact on other neurodegenerative diseases: Importantly, the physical infrastructure and knowledge gained from this ERC project have enabled us to extend our synthetic capabilities to addressing the role of post-translational modifications in other important neurodegenerative diseases, including Alzheimer’s and Huntington’s disease. We recently reported the first one-pot Semisynthesis of WT and phosphorylated forms untagged forms of exon1 of the Huntingtin protein. These studies revealed novel observations that were masked by the use of constructs on the basis of using fusion proteins.
